Isolation Of Microbes And Their Acid-producing Characteristics In The Solid-cultures Of Zhenjiang Vinegar

Zhenjiang Aromatic Vinegar is one of the four Chinese famous vinegars. The vinegar industry belongs to traditional fermentation techniques. The acetic acid fermentation of Zhenjiang vinegar lasted about twenty days, and the 7th-day's cultures were used for inoculation, so the microbial structure and quality of each fermentation circle were comparatively stable.The dynamic changes of acid-producing microbes and fungi were studied here during the acetic-acid fermentation process. The results revealed that acid-producing microbes, at 7th-day's solid cultures of Zhenjiang vinegar, mounted to their summit populations while descended after that. As to fungi, they decreased in the whole course of acetic-acid fermentation.Acid-producing microbes and fungi isolated from the solid cultures of zhengjiang vinegar were identified by molecular techniques. First the 16S rDNA or 18S rDNA were amplified, then the PCR products were clustered into different OTUs according to the ARDRA types with restriction enzymes Hinf I and Csp6 I, and then every OTU was sequenced, subjected to NCBI and blasted to determine their specific genera or species. The results showed that five sorts of acid-producing microbes were identified, which were Staphylococcus sp., Bacillus ruris, Acetobacter pasteurianus, Lactobacillus casei and Paenibacillus sp., respectively. But only one kind of yeast, that is Candida rugosa, was found here. Among these five acid-producing microbes Staphylococcus sp. was isolated from solid cultures of vinegar for the first time, and Bacillus ruris, Paenibacillus sp., as acid-producing bacillus, were also first isolated in solid cultures of vinegar. Their performances in the vinegar-brewing were not well investigated and should be taken into further studies.After that the five identified acid-producing microbes were cultivated in shaking flasks, and the organic acids composition (oxalic acid, tartaric acid, pyruvic acid, malic acid, oxaglutaric acid, lactic acid, acetic acid, citric acid, fumaric acid and succinic acid)were extracted and tested by HPLC, in the purpose of investigating their performances in the brewing of Zhenjiang vinegar. The results revealed that all of them can produce lactic acid which was the greatest unvolatile component in Zhenjiang vinegar, so most probably that all these different acid-producing microbes were one of the real reasons yielding high lactic-acid quantities in Zhenjiang vinegar. Additionally seven kind of organic acids were also detected in this experiment. So we can come to the conclusion that the affluent organic acid composition produced by these different acid-producing microbes will greatly improve the flavor of Zhenjiang aromatic vinegar. And we also studied the organic acid produced by solid cultures of Bacillus ruris, which was quite different from that of its liquid fermentation broth.